RFRP-3在湖羊雄性生殖器官中的表達及其受營養(yǎng)水平的影響
發(fā)布時間：2018-12-18 05:36
【摘要】：湖羊作為一個高繁殖力綿羊品種,目前的研究主要集中在雌性湖羊的繁殖生理,而對雄性湖羊的繁殖生理關(guān)注較少。2000年,日本科學(xué)家Tsutsui等從日本鵪鶉腦內(nèi)成功分離提純一種RF酰胺肽,進一步分析發(fā)現(xiàn),其為C末端具有RF酰胺的十二肽,而且對鳥類垂體前葉GnRH的分泌有抑制作用,故命名為促性腺激素抑制激素(Gonadotropin-inhibitory hormone,GnIH)。RF 酰胺相關(guān)肽-3(RFamide-related peptide-3,RFRP-3)是GnIH在哺乳動物體內(nèi)的同系物,它可以調(diào)節(jié)哺乳動物的繁殖、攝食、焦慮、應(yīng)激和能量平衡等行為,是目前生殖生物學(xué)領(lǐng)域研究的熱點之一,已在多種動物的不同組織均有表達,但其在雄性湖羊生殖系統(tǒng)中的表達情況還不清楚。本試驗主要利用熒光定量PCR、免疫組化等技術(shù),以10日齡、4月齡和8月齡的雄性湖羊的睪丸、附睪頭、附睪體、附睪尾和輸精管為研究對象,檢測RFRP-3在其發(fā)育過程中各組織的定位及表達情況;再以6月齡湖羊雄性生殖器官為研究對象,分析不同營養(yǎng)水平飼喂下,RFRP-3、GnRHR、FSHR和LHR基因在雄性湖羊生殖器官的表達變化,旨在研究分析RFRP-3在雄性湖羊生殖器官生長發(fā)育過程中的作用,以及其參與繁殖營養(yǎng)調(diào)控的作用。主要的研究結(jié)果如下:1.RFRP-3蛋白在湖羊雄性生殖器官發(fā)育過程中的定位研究。RFRP-3蛋白主要分布于8月齡湖羊睪丸的生精小管內(nèi)部、輸精管及附睪;RFRP-3蛋白在8月齡湖羊附睪的假復(fù)層柱狀上皮和輸精管的粘膜層中強烈表達,而在4月齡和10日齡湖羊生殖器官中微弱表達。2.RFRP-3基因在湖羊雄性生殖器官發(fā)育過程中的定量研究。在10日齡湖羊組中,RFRP-3基因在附睪頭、附睪體、附睪尾、睪丸和輸精管組織中表達量差異不顯著。在4月齡和8月齡湖羊組中,RFRP-3基因在附睪頭、附睪體、附睪尾和睪丸的表達量差異不顯著,而在輸精管中的表達量顯著高于其他組織(P0.05)。此外,在同一月齡不同組織中RFRP-3基因mRNA表達水平也存在一定的差異。3.不同營養(yǎng)水平下對湖羊雄性生殖器官中RFRP-3、GnRHR、FSHR和LHR基因mRNA表達的影響。在高能高蛋白水平飼喂下,RFRP-3基因在雄性湖羊生殖器官中表達量顯著低于正常飼喂水平,而GnRHR、FS和LHR基因mRNA表達量顯著高于正常飼喂水平。以上試驗結(jié)果證明,RFRP-3在湖羊雄性生殖器官中廣泛分布,其表達量在湖羊不同生殖器官、不同月齡和不同飼喂水平條件下差異顯著。本研究為進一步探討RFRP-3的生物功能以及其參與動物繁殖營養(yǎng)調(diào)控研究提供了理論依據(jù),但其具體的調(diào)節(jié)機制以及與其它調(diào)節(jié)因子之間的相互作用還需要進一步的研究。
[Abstract]:As a high fecundity sheep breed, the current research focuses on the reproductive physiology of female Hu sheep, but less attention on the reproductive physiology of male Hu sheep. Japanese scientist Tsutsui et al successfully isolated and purified a RF amide peptide from the brain of Japanese quail. Further analysis showed that it was a dodeceptide with RF amide at the C-terminal and inhibited the secretion of GnRH in the anterior pituitary of birds. Therefore, gonadotropin inhibitory hormone (RFamide-related peptide-3,RFRP-3) is a homologue of GnIH in mammals, which can regulate mammalian reproduction, feeding and anxiety. The behaviors of stress and energy balance are one of the hotspots in the field of reproductive biology. They have been expressed in different tissues of many animals, but their expression in the reproductive system of male Hu sheep is still unclear. In this study, the testis, head of epididymis, body of epididymis, tail of epididymis and vas deferens of 10 days old, 4 months old and 8 months old male Hu sheep were studied by fluorescence quantitative PCR, immunohistochemical technique. The localization and expression of RFRP-3 in various tissues during its development were detected. The expression of RFRP-3,GnRHR,FSHR and LHR genes in male reproductive organs of six month old Hu sheep was analyzed under different nutrition levels. The purpose of this study was to investigate the role of RFRP-3 in the growth and development of reproductive organs of male Hu sheep and its role in reproductive and nutritional regulation. The main results are as follows: the localization of 1.RFRP-3 protein in the development of male reproductive organs in Hu sheep. The RFRP-3 protein was mainly distributed in the seminiferous tubules, vas deferens and epididymis of 8 months old Hu sheep testis. RFRP-3 protein was strongly expressed in the pseudostratified columnar epithelium of the epididymis and the mucosal layer of the vas deferens in the 8-month-old Hu sheep epididymis. The expression of 2.RFRP-3 gene in male reproductive organs of Hu sheep at the age of 4 months and 10 days was studied quantitatively. There was no significant difference in the expression of RFRP-3 gene in the head of epididymis, corpus epididymidis, cauda epididymidis, testis and vas deferens in 10-day-old Hu sheep group. The expression of RFRP-3 gene in the head of epididymis, body of epididymis, cauda epididymidis and testis was not significantly different in 4 and 8 months old Hu sheep group, but the expression level in vas deferens was significantly higher than that in other tissues (P0.05). In addition, the mRNA expression level of RFRP-3 gene was also different in different tissues of the same month. 3. 3. Effects of different nutritional levels on mRNA expression of RFRP-3,GnRHR,FSHR and LHR genes in male reproductive organs of Hu sheep. Under high energy and high protein levels, the expression of RFRP-3 gene in male Hu sheep reproductive organs was significantly lower than that in normal feeding, while the mRNA expression of GnRHR,FS and LHR genes was significantly higher than that of normal feeding. The results showed that RFRP-3 was widely distributed in male reproductive organs of Hu sheep, and the expression of RFRP-3 was significantly different in different reproductive organs, different months of age and different feeding levels of Hu sheep. This study provides a theoretical basis for further study on the biological function of RFRP-3 and its involvement in animal reproductive and nutritional regulation, but its specific regulation mechanism and interaction with other regulatory factors need to be further studied.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S826
本文編號：2385429
[Abstract]:As a high fecundity sheep breed, the current research focuses on the reproductive physiology of female Hu sheep, but less attention on the reproductive physiology of male Hu sheep. Japanese scientist Tsutsui et al successfully isolated and purified a RF amide peptide from the brain of Japanese quail. Further analysis showed that it was a dodeceptide with RF amide at the C-terminal and inhibited the secretion of GnRH in the anterior pituitary of birds. Therefore, gonadotropin inhibitory hormone (RFamide-related peptide-3,RFRP-3) is a homologue of GnIH in mammals, which can regulate mammalian reproduction, feeding and anxiety. The behaviors of stress and energy balance are one of the hotspots in the field of reproductive biology. They have been expressed in different tissues of many animals, but their expression in the reproductive system of male Hu sheep is still unclear. In this study, the testis, head of epididymis, body of epididymis, tail of epididymis and vas deferens of 10 days old, 4 months old and 8 months old male Hu sheep were studied by fluorescence quantitative PCR, immunohistochemical technique. The localization and expression of RFRP-3 in various tissues during its development were detected. The expression of RFRP-3,GnRHR,FSHR and LHR genes in male reproductive organs of six month old Hu sheep was analyzed under different nutrition levels. The purpose of this study was to investigate the role of RFRP-3 in the growth and development of reproductive organs of male Hu sheep and its role in reproductive and nutritional regulation. The main results are as follows: the localization of 1.RFRP-3 protein in the development of male reproductive organs in Hu sheep. The RFRP-3 protein was mainly distributed in the seminiferous tubules, vas deferens and epididymis of 8 months old Hu sheep testis. RFRP-3 protein was strongly expressed in the pseudostratified columnar epithelium of the epididymis and the mucosal layer of the vas deferens in the 8-month-old Hu sheep epididymis. The expression of 2.RFRP-3 gene in male reproductive organs of Hu sheep at the age of 4 months and 10 days was studied quantitatively. There was no significant difference in the expression of RFRP-3 gene in the head of epididymis, corpus epididymidis, cauda epididymidis, testis and vas deferens in 10-day-old Hu sheep group. The expression of RFRP-3 gene in the head of epididymis, body of epididymis, cauda epididymidis and testis was not significantly different in 4 and 8 months old Hu sheep group, but the expression level in vas deferens was significantly higher than that in other tissues (P0.05). In addition, the mRNA expression level of RFRP-3 gene was also different in different tissues of the same month. 3. 3. Effects of different nutritional levels on mRNA expression of RFRP-3,GnRHR,FSHR and LHR genes in male reproductive organs of Hu sheep. Under high energy and high protein levels, the expression of RFRP-3 gene in male Hu sheep reproductive organs was significantly lower than that in normal feeding, while the mRNA expression of GnRHR,FS and LHR genes was significantly higher than that of normal feeding. The results showed that RFRP-3 was widely distributed in male reproductive organs of Hu sheep, and the expression of RFRP-3 was significantly different in different reproductive organs, different months of age and different feeding levels of Hu sheep. This study provides a theoretical basis for further study on the biological function of RFRP-3 and its involvement in animal reproductive and nutritional regulation, but its specific regulation mechanism and interaction with other regulatory factors need to be further studied.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S826
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